Oligonucleotides are provided which are targeted to nucleic acids encoding human c-raf and capable of inhibiting raf expression. The oligonucleotides contain a methoxyethoxy (2xe2x80x2xe2x80x94Oxe2x80x94CH2CH2OCH3) modification at the 2xe2x80x2 position of at least one nucleotide. Methods of inhibiting the expression of human raf using oligonucleotides of the invention are also provided. The present invention further comprises methods of inhibiting hyperproliferation of cells and methods of treating abnormal proliferative conditions which employ oligonucleotides of the invention.

This invention relates to compositions and methods for modulating expression of the raf gene, a naturally present cellular gene which has been implicated in abnormal cell proliferation and tumor formation. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the raf gene.
Alterations in the cellular genes which directly or indirectly control cell growth and differentiation are considered to be the main cause of cancer. The raf gene family includes three highly conserved genes termed A-, B- and c-raf (also called raf-1). Raf genes encode protein kinases that are thought to play important regulatory roles in signal transduction processes that regulate cell proliferation. Expression of the c-raf protein is believed to play a role in abnormal cell proliferation since it has been reported that 60% of all lung carcinoma cell lines express unusually high levels of c-raf mRNA and protein. Rapp et al., The Oncogene Handbook, E. P. Reddy, A. M Skalka and T. Curran, eds., Elsevier Science Publishers, New York, 1988, pp. 213-253.
Oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. For example, workers in the field have now identified antisense, triplex and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases.
As examples, U.S. Pat. No. 5,135,917, issued Aug. 4, 1992, provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression. U.S. Pat. No. 5,098,890, issued Mar. 24, 1992 in the name of Gewirtz et al., is directed to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapies for certain cancerous conditions. U.S. Pat. No. 5,087,617, issued Feb. 11, 1992, provides methods for treating cancer patients with antisense oligonucleotides. U.S. Pat. No. 5,166,195 issued Nov. 24, 1992, provides oligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810, issued Apr. 2, 1991, provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428, issued Mar. 16, 1993, provides antisense oligonucleotides having antiviral activity against influenza virus. U.S. Pat. No. 4,806,463, issued Feb. 21, 1989, provides antisense oligonucleotides and methods using them to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717 (Cohen et al.), issued Feb. 15, 1994, is directed to a mixed linkage oligonucleotide phosphorothioates complementary to an oncogene; U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 (Cohen et al.) are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells. Antisense oligonucleotides have been safely administered to humans and clinical trials of several antisense oligonucleotide drugs, targeted both to viral and cellular gene products, are presently underway. The phosphorothioate oligonucleotide, ISIS 2922, has been shown to be effective against cytomegalovirus retinitis in AIDS patients. BioWorld Today, Apr. 29, 1994, p. 3. It is thus established that oligonucleotides can be useful therapeutic instrumentalities and can be configured to be useful in treatment regimes for treatment of cells and animal subjects, especially humans.
Antisense oligonucleotide inhibition of gene expression has proven to be a useful tool in understanding the roles of raf genes. An antisense oligonucleotide complementary to the first six codons of human c-raf has been used to demonstrate that the mitogenic response of T cells to interleukin-2 (IL-2) requires c-raf. Cells treated with the oligonucleotide showed a near-total loss of c-raf protein and a substantial reduction in proliferative response to IL-2. Riedel et al., Eur. J. Immunol. 1993, 23, 3146-3150. Rapp et al. have disclosed expression vectors containing a raf gene in an antisense orientation downstream of a promoter, and methods of inhibiting raf expression by expressing an antisense Raf gene or a mutated Raf gene in a cell. WO application 93/04170. An antisense oligodeoxyribonucleotide complementary to codons 1-6 of murine c-Raf has been used to abolish insulin stimulation of DNA synthesis in the rat hepatoma cell line H4IIE. Tornkvist et al., J. Biol. Chem. 1994, 269, 13919-13921. WO Application 93/06248 discloses methods for identifying an individual at increased risk of developing cancer and for determining a prognosis and proper treatment of patients afflicted with cancer comprising amplifying a region of the c-raf gene and analyzing it for evidence of mutation.
Denner et al. disclose antisense polynucleotides hybridizing to the gene for raf, and processes using them. WO 94/15645. Oligonucleotides hybridizing to human and rat raf sequences are disclosed.
Iversen et al. disclose heterotypic antisense oligonucleotides complementary to raf which are able to kill ras-activated cancer cells, and methods of killing raf-activated cancer cells. Numerous oligonucleotide sequences are disclosed, none of which are actually antisense oligonucleotide sequences.
There remains a long-felt need for improved compositions and methods for inhibiting raf gene expression.
The present invention provides oligonucleotides which are targeted to nucleic acids encoding human raf and are capable of inhibiting raf expression. The oligonucleotides contain a methoxyethoxy (2xe2x80x2xe2x80x94Oxe2x80x94CH2CH2OCH3) modification at the 2xe2x80x2 position of at least one nucleotide. These oligonucleotides are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.
The present invention also comprises methods of inhibiting the expression of human raf, particularly the abnormal expression of raf, using the oligonucleotides of the invention. These methods are believed to be useful both therapeutically and diagnostically as a consequence of the association between raf expression and hyperproliferation. These methods are also useful as tools, for example for detecting and determining the role of raf expression in various cell functions and physiological processes and conditions and for diagnosing conditions associated with raf expression.
The present invention also comprises methods of inhibiting hyperproliferation of cells using oligonucleotides of the invention. These methods are believed to be useful, for example in diagnosing raf-associated cell hyperproliferation. Methods of treating abnormal proliferative conditions are also provided. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically and as clinical research and diagnostic tools.
Malignant tumors develop through a series of stepwise, progressive changes that lead to the loss of growth control characteristic of cancer cells, i.e., continuous unregulated proliferation, the ability to invade surrounding tissues, and the ability to metastasize to different organ sites. Carefully controlled in vitro studies have helped define the factors that characterize the growth of normal and neoplastic cells and have led to the identification of specific proteins that control cell growth and differentiation. The raf genes are members of a gene family which encode related proteins termed A-, B- and c-raf. Raf genes code for highly conserved serine-threonine-specific protein kinases. These enzymes are differentially expressed; c-raf, the most thoroughly characterized, is expressed in all organs and in all cell lines that have been examined. A- and B-raf are expressed in urogenital and brain tissues, respectively. c-raf protein kinase activity and subcellular distribution are regulated by mitogens via phosphorylation. Various growth factors, including epidermal growth factor, acidic fibroblast growth factor, platelet-derived growth factor, insulin, granulocyte-macrophage colony-stimulating factor, interleukin-2, interleukin-3 and erythropoietin, have been shown to induce phosphorylation of c-raf. Thus, c-raf is believed to play a fundamental role in the normal cellular signal transduction pathway, coupling a multitude of growth factors to their net effect, cellular proliferation.
Certain abnormal proliferative conditions are believed to be associated with raf expression and are, therefore, believed to be responsive to inhibition of raf expression. Abnormally high levels of expression of the raf protein are also implicated in transformation and abnormal cell proliferation. These abnormal proliferative conditions are also believed to be responsive to inhibition of raf expression. Examples of abnormal proliferative conditions are hyperproliferative disorders such as cancers, tumors, hyperplasias, pulmonary fibrosis, angiogenesis, psoriasis, atherosclerosis and smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. The cellular signaling pathway of which raf is a part has also been implicated in inflammatory disorders characterized by T-cell proliferation (T-cell activation and growth), such as tissue graft rejection, endotoxin shock, and glomerular nephritis, for example.
It has now been found that elimination or reduction of raf gene expression can halt or reverse abnormal cell proliferation. This has been found even when levels of raf expression are not abnormally high. There is a great desire to provide compositions of matter which can modulate the expression of the raf gene. It is greatly desired to provide methods of detection of the raf gene in cells, tissues and animals. It is also desired to provide methods of diagnosis and treatment of abnormal proliferative conditions associated with abnormal raf gene expression. In addition, kits and reagents for detection and study of the raf gene are desired. xe2x80x9cAbnormalxe2x80x9d raf gene expression is defined herein as abnormally high levels of expression of the raf protein, or any level of raf expression in an abnormal proliferative condition or state.
The present invention employs oligonucleotides targeted to nucleic acids encoding raf. This relationship between an oligonucleotide and its complementary nucleic acid target to which it hybridizes is commonly referred to as xe2x80x9cantisensexe2x80x9d. xe2x80x9cTargetingxe2x80x9d an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the target is a nucleic acid encoding raf; in other words, the raf gene or mRNA expressed from the raf gene. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the oligonucleotide interaction to occur such that the desired effectxe2x80x94modulation of gene expressionxe2x80x94will result. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.
In the context of this invention xe2x80x9cmodulationxe2x80x9d means either inhibition or stimulation. Inhibition of raf gene expression is presently the preferred form of modulation. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression or Western blot assay of protein expression as taught in the examples of the instant application. Effects on cell proliferation or tumor cell growth can also be measured, as taught in the examples of the instant application. xe2x80x9cHybridizationxe2x80x9d, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them. xe2x80x9cSpecifically hybridizablexe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
In the present invention, oligonucleotides are provided which are targeted to mRNA encoding c-raf. In accordance with this invention, persons of ordinary skill in the art will understand that mRNA includes not only the coding region which carries the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5xe2x80x2-untranslated region, the 3xe2x80x2-untranslated region, the 5xe2x80x2 cap region, intron regions and intron/exon or splice junction ribonucleotides. Thus, oligonucleotides may be formulated which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides. The functions of messenger RNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing or maturation of the RNA and possibly even independent catalytic activity which may be engaged in by the RNA. The overall effect of such interference with the RNA function is to cause interference with raf protein expression.
The present invention provides oligonucleotides for modulation of raf gene expression. Such oligonucleotides are targeted to nucleic acids encoding c-raf.
In the context of this invention, the term xe2x80x9coligonucleotidexe2x80x9d refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term xe2x80x9coligonucleotidexe2x80x9d also includes oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Modifications may be on one or more bases, sugars, or backbone linkages, or combinations of these; such modifications are well known in the art. Modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
The oligonucleotides may be chimeric oligonucleotides. xe2x80x9cChimeric oligonucleotidesxe2x80x9d or xe2x80x9cchimerasxe2x80x9d, in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the RNA target) and a region that is a substrate for RNase H cleavage. In one embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAse H. Affinity of an oligonucleotide for its target (in this case a nucleic acid encoding raf) is routinely determined by measuring the Tm of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonucleotide for the target.
Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2xe2x80x2-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance antisense oligonucleotide inhibition of raf gene expression. RNAse H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of antisense inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.
The oligonucleotides of the present invention contain a methoxyethoxy (2xe2x80x2xe2x80x94Oxe2x80x94CH2CH2OCH3) modification at the 2xe2x80x2 position of at least one nucleotide. This modification has been shown to increase both affinity of the oligo nucleotide for its target and nuclease resistance of the oligonucleotide. Oligonucleotides in accordance with this invention are preferably from about 8 to about 50 nucleotides in length. In the context of this invention it is understood that this encompasses non-naturally occurring oligomers as hereinbefore described, having 8 to 50 monomers.
The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Martin, P., Helv. Chim. Acta 1995, 78,486-504. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling Va.) to synthesize fluorescently labeled, biotinylated or other conjugated oligonucleotides.
It has now been found that certain oligonucleotides targeted to portions of the c-raf mRNA are particularly useful for inhibiting raf expression and for interfering with cell hyperproliferation. Methods for inhibiting c-raf expression using antisense oligonucleotides are, likewise, useful for interfering with cell hyperproliferation. In the methods of the invention, tissues or cells are contacted with oligonucleotides. In the context of this invention, to xe2x80x9ccontactxe2x80x9d tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal.
For therapeutics, methods of inhibiting hyperproliferation of cells and methods of treating abnormal proliferative conditions are provided. The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill in the art. In general, for therapeutics, a patient suspected of needing such therapy is given an oligonucleotide in accordance with the invention, commonly in a pharmaceutically acceptable carrier, in amounts and for periods which will vary depending upon the nature of the particular disease, its severity and the patient""s overall condition. The pharmaceutical compositions of this invention may be administered in a number of ways depending upon whether local or systemic treatment is desired, and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, or parenteral, for example by intravenous drip, intravenous injection or subcutaneous, intraperitoneal or intramuscular injection.
Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
In addition to such pharmaceutical carriers, cationic lipids may be included in the formulation to facilitate oligonucleotide uptake. One such composition shown to facilitate uptake is Lipofectin (BRL, Bethesda MD).
Dosing is dependent on severity and responsiveness of the condition to be treated, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be calculated based on EC50""s in in vitro and in vivo animal studies. For example, given the molecular weight of compound (derived from oligonucleotide sequence and chemical structure) and an effective dose such as an IC50, for example (derived experimentally), a dose in mg/kg is routinely calculated.
The present invention is also suitable for diagnosing abnormal proliferative states in tissue or other samples from patients suspected of having a hyperproliferative disease such as cancer, psoriasis or blood vessel restenosis or atherosclerosis. The ability of the oligonucleotides of the present invention to inhibit cell proliferation may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. Similarly, the present invention can be used to distinguish raf-associated tumors from tumors having other etiologies, in order that an efficacious treatment regime can be designed.
The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.
The oligonucleotides of the invention are also useful for detection and diagnosis of raf expression. For example, radiolabeled oligonucleotides can be prepared by 32p labeling at the 5xe2x80x2 end with polynucleotide kinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, p. 10.59. Radiolabeled oligonucleotides are then contacted with tissue or cell samples suspected of raf expression and the sample is washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates bound oligonucleotide (which in turn indicates the presence of raf) and can be quantitated using a scintillation counter or other routine means. Radiolabeled oligo can also be used to perform autoradiography of tissues to determine the localization, distribution and quantitation of raf expression for research, diagnostic or therapeutic purposes. In such studies, tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to routine autoradiography procedures. The emulsion, when developed, yields an image of silver grains over the regions expressing raf. Quantitation of the silver grains permits raf expression to be detected.
Analogous assays for fluorescent detection of raf expression can be developed using oligonucleotides of the invention which are conjugated with fluorescein or other fluorescent tag instead of radiolabeling. Such conjugations are routinely accomplished during solid phase synthesis using fluorescently labeled amidites or CPG (e.g., fluorescein-labeled amidites and CPG available from Glen Research, Sterling Va. See 1993 Catalog of Products for DNA Research, Glen Research, Sterling Va., p. 21).
Each of these assay formats is known in the art. One of skill could easily adapt these known assays for detection of raf expression in accordance with the teachings of the invention providing a novel and useful means to detect raf expression.
Phosphorothioate deoxyoligonucleotides targeted to human c-raf were designed using the Genbank c-raf sequence HUMRAFR (Genbank listing xc3x9703484), synthesized and tested for inhibition of c-raf mRNA expression in T24 bladder carcinoma cells using a Northern blot assay. Oligonucleotide ISIS 5132 (TCCCGCCTGTGACATGCATT; SEQ ID NO: 1), targeted to the 3xe2x80x2UTR of c-raf, showed greater than 90% inhibition.
Two oligonucleotides having SEQ ID NO: 1 and an 8-nucleotide deoxynucleotide central region flanked on each side by 6 nucleotides having the 2xe2x80x2xe2x80x94Oxe2x80x94CH2CH2OCH3 modification were synthesized. These compounds differ in that one of them, ISIS 10755 (also known as CIBA 1440) has a uniform phosphorothioate backbone; the other, ISIS 10754 (also known as CIBA 1439 or CGP 69845) has a phosphorothioate backbone in the central region (backbone linkages 7-14) and a phosphodiester backbone in the remaining (flanking) regions. These oligonucleotides were tested for their ability to inhibit c-raf mRNA expression in T24 cells. IC50s were calculated (oligonucleotide concentration yielding 50% inhibition) and are shown in Table 1 along with Tm data showing affinity of these oligonucleotides for their complement. Because of their extremely low IC50s, both ISIS 10755 and ISIS 10754 are preferred.
Specificity of ISIS 5132 for raf mRNA was demonstrated by a Northern blot assay in which this oligonucleotide was tested for the ability to inhibit Ha-ras mRNA as well as c-raf mRNA in T24 cells. Ha-ras is a cellular oncogene which is implicated in transformation and tumorigenesis. ISIS 5132 was shown to abolish c-raf MRNA almost completely with no effect on Ha-ras mRNA levels.
Chimeric oligonucleotides having SEQ ID NO: 1 were prepared. These oligonucleotides had central xe2x80x9cgapxe2x80x9d regions of 6, 8, or 10 deoxynucleotides flanked by two regions of 2xe2x80x2-O-methyl modified nucleotides. Backbones were uniformly phosphorothioate. In Northern blot analysis, all three of these oligonucleotides (ISIS 6720, 6-deoxy gap; ISIS 6717, 8-deoxy gap; ISIS 6729, 10-deoxy gap) showed greater than 70% inhibition of c-raf mRNA expression in T24 cells. The 8-deoxy gap compound (6717) showed greater than 90% inhibition. Oligonucleotides of SEQ ID NO: 1 having an 8-deoxynucleotide gap flanked by either 2xe2x80x2-O-propyl or 2xe2x80x2 fluoro modified nucleotides were also found to be active.
The phosphorothioate oligonucleotide ISIS 5132 (SEQ ID NO: 1) was shown to inhibit T24 bladder cancer cell proliferation. Cells were treated with various concentrations of oligonucleotide in conjunction with lipofectin (cationic lipid which increases uptake of oligonucleotide). A dose-dependent inhibition of cell proliferation was demonstrated, as indicated in Table 2, in which xe2x80x9cNonexe2x80x9d indicates untreated control (no oligonucleotide) and xe2x80x9cControlxe2x80x9d indicates treatment with negative control oligonucleotide. Results are shown as percent inhibition compared to untreated control.
Subcutaneous human T24 bladder carcinoma xenografts in nude mice were established and treated with ISIS 5132 and an unrelated control phosphorothioate oligonucleotide administered intraperitoneally three times weekly at a dosage of 25 mg/kg. In this preliminary study, ISIS 5132 inhibited tumor growth after eleven days by 35% compared to controls. Oligonucleotide-treated tumors remained smaller than control tumors throughout the course of the study.
Many conditions which are believed to be mediated by raf kinase are not amenable to study in humans. For example, tissue graft rejection is a condition which is likely to be ameliorated by interference with raf expression; but, clearly, this must be evaluated in animals rather than human transplant patients. Another such example is restenosis. These conditions can be tested in animal models, however, such as the rat and mouse models used here. These data provide added evidence of the therapeutic utility of antisense oligonucleotides targeted to c-raf.
Oligonucleotide sequences for inhibiting c-raf expression in rat and mouse cells were identified. Rat and mouse c-raf genes have regions of high homology; a series of oligonucleotides which target both rat and mouse c-raf mRNA sequence were designed and synthesized, using information gained from evaluation of oligonucleotides targeted to human c-raf. These oligonucleotides were screened for activity in mouse bEND cells and rat A-10 cells using Northern blot assays. Two oligonucleotides, ISIS 11061 and ISIS 10707, were found to inhibit c-raf RNA levels by greater than 90% in mouse bEND cells at a dose of 400 nM. These two oligonucleotides inhibited raf RNA levels virtually entirely in rat A-10 cells at a concentration of 200 nM. The IC50 for ISIS 10707 was found to be 170 nM in mouse bEND cells and 85 nM in rat A-10 cells. The IC50 for ISIS 11061 was determined to be 85 nM in mouse bEND cells and 30 nM in rat A-10 cells.
Effect of ISIS 11061 on Endogenous c-raf mRNA Expression in Mice
Mice were injected intraperitoneally with ISIS 11061 (50 mg/kg) or control oligonucleotide or saline control once daily for three days. Animals were sacrificed and organs were analyzed for c-raf mRNA expression by Northern blot analysis. ISIS 11061 was found to decrease levels of c-raf mRNA in liver by approximately 70%. Control oligonucleotides had no effects on c-raf expression. The effect of ISIS 11061 was specific for c-raf; A-raf and G3PDH RNA levels were unaffected by oligonucleotide treatment.
To determine the therapeutic effects of the c-raf antisense oligonucleotide ISIS 11061 in preventing allograft rejection, this oligonucleotide was tested for activity in a murine vascularized heterotopic heart transplant model. Hearts from C57BI10 mice were transplanted into the abdominal cavity of C3H mice as primary vascularized grafts essentially as described by Isobe et al., Circulation 1991, 84, 1246-1255. Oligonucleotides were administered by continuous intravenous administration via a 7-day Alzet pump. The mean allograft survival time for untreated mice was 7.83+0.75 days(7, 7, 8, 8, 8, 9 days). Allografts in mice treated for 7 days with 20 mg/kg or 40 mg/kg ISIS 11061 all survived at least 11 days (11, 11, 12 days for 20 mg/kg dose and  greater than 11,  greater than 11,  greater than 11 days for the 40 mg/kg dose).
In a pilot study conducted in rats, hearts from Lewis rats were transplanted into the abdominal cavity of ACI rats. Rats were dosed with ISIS 11061 at 20 mg/kg for 7 days via Alzet pump. The mean allograft survival time for untreated rats was 8.86+0.69 days (8, 8, 9, 9, 9, 9, 10 days). In rats treated with oligonucleotide, the allograft survival time was 15.3xc2x11.15 days (14, 16, 16 days).
Smooth muscle cell proliferation is a cause of blood vessel stenosis, for example in atherosclerosis and restenosis after angioplasty. Experiments were performed to determine the effect of ISIS 11061 on proliferation of A-10 rat smooth muscle cells. Cells in culture were grown with and without ISIS 11061 (plus lipofectin) and cell proliferation was measured 24 and 48 hours after stimulation with fetal calf serum. ISIS 11061 (500 nM) was found to inhibit serum-stimulated cell growth in a dose-dependent manner with a maximal inhibition of 46% and 75% at 24 hours and 48 hours, respectively. An IC50 value of 200 nM was obtained for this compound. An unrelated control oligonucleotide had no effect at doses up to 500 nM.
A rat carotid artery injury model of angioplasty restenosis has been developed and has been used to evaluate the effects on restenosis of antisense oligonucleotides targeted to the c-myc oncogene. Bennett et al., J. Clin. Invest. 1994, 93, 820-828. This model will be used to evaluate the effects of antisense oligonucleotides targeted to rat c-raf, particularly ISIS 11061, on restenosis. Following carotid artery injury with a balloon catheter, oligonucleotides are administered either by intravenous injection, continuous intravenous administration via Alzet pump, or direct administration to the carotid artery in a pluronic gel matrix as described by Bennett et al. After recovery, rats are sacrificed, carotid arteries are examined by microscopy and effects of treatment on luminal cross-sections are determined.
The invention is further illustrated by the following examples which are illustrations only and are not intended to limit the present invention to specific embodiments.

Antisense oligonucleotide inhibition of raf gene expression


A modular doll house has interconnectable walls, floors and roofs that are held together by a variety of connectors. The connectors of the modular doll house include tabs which slidably engage the interconnectable walls, floors and roofs and can be easily manipulated by children to assemble and disassemble the modular doll house. Specifically, the modular doll house includes a first structural member having a first surface and a first groove disposed in the first surface and a second structural member having a second surface and a second groove disposed in the second surface. Additionally, the modular doll house has a rigid connector including a first tab adapted to slidably engage the first groove and a second tab adapted to slidably engage the second groove to connect the first structural member to the second structural member.

The present invention relates generally to doll houses, and more particularly, to connector systems used to construct modular doll houses having interconnectable walls, floors and roofs.
It is common in the marketplace to find pre-assembled, permanently constructed doll houses. However, such doll houses are difficult to ship and store. Additionally, pre-assembled doll houses are often expensive and, thus, not affordable because of the manufacturer""s high labor costs, which drive up the overall cost of the doll houses.
To avoid some of the problems associated with the pre-assembled, permanently constructed doll houses, manufacturers have created modular doll houses having interconnectable walls, floors and roofs that require self-assembly. Because modular doll houses can be constructed and/or taken apart, the interconnectable doll house parts have eliminated several problems associated with shipping, storage and cost. Despite such improvements, these known modular doll houses still have disadvantages. For example, most modular doll houses are made of specially designed walls, floors, roofs and other structural accessories such as fireplaces, stairs and railings which, when constructed, form a specific doll house configuration having a single, predetermined floor plan. This prefabricated configuration stifles a child""s imagination and creativity. Additionally, modular doll houses are often difficult to assemble and usually require special building materials and tools. Furthermore, the walls of these modular doll houses are usually pre-cut to a certain size, which is often proportional to the size of a traditional doll house figure standing approximately 3-4 inches in height. Thus, children having these specially configured modular doll houses cannot use them with larger dolls such as Barbie(copyright) dolls, because these modular doll houses are too small.
Other modular doll houses require special connectors including pegs or pins to hold the panels together. For example, panels are held together by inserting pegs into holes in the panels. Because these special connectors include many small parts, they are difficult for young children to assemble. Moreover, the connectors used in typical modular doll houses lack strength and durability and, therefore, these modular doll houses lack the stability needed to withstand every day wear and tear.
U.S. Pat. No. 5,647,181 to Hunts discloses a method of creating a doll house using rigid sheet-like panels and panel connectors which operate to join the panels together. In operation, the connectors accept specifically configured edges of the sheet-like panels to form a locking, yet releasable snap-fit connection. The components of this system enable panels to be joined together in different arrangements to form building structures, like doll houses, with a variety of rooms, levels and floor plans.
A shortcoming of the connector system disclosed in the Hunts patent is that the panels must be cut to a predetermined dimension to create the specially configured panel edges. Such pre-cut panels hamper the creativity of the child and limit the various arrangements of panels for the doll house. Furthermore, specially configured connectors complicate manufacturing, as well as assembly.
U.S. Pat. Nos. 3,906,659 to Walmer, 4,306,371 to Walmer et al. and 4,219,960 to Walmer et al. (hereinafter xe2x80x9cthe Walmer patentsxe2x80x9d) disclose collapsible doll houses that are constructed of a small number of individual panels making up the walls, floors and roofs of the doll houses. The panels are provided with grooves and slots so that all the panels slide together and support one another. Likewise, a number of different connector elements may be used to interconnect the edges of the walls, floors and roofs of these doll houses.
Although the Walmer patents provide an improved method for self assembly of doll houses, they disadvantageously restrict the doll houses to a pre-arranged, specified number of rooms that are limited in size. Also, the doll houses disclosed in these patents stifle the creativity of a child and preclude the child from playing with dolls of different sizes, especially dolls which are larger than traditional dolls.
The modular doll house according to the present invention has interconnectable structural members such as walls, floors and roofs that are held together by a variety of connectors. The connectors include tabs which slidably engage grooves cut into the structural members and, thus, join the structural members together. The walls, floors and roofs are interconnectable, in any number of configurations and thus, the modular doll house can be assembled in many different ways to stimulate a child""s creativity. Additionally, the interconnectable structural members may be purchased pre-cut or may be purchased in large sheet-like panels which may be cut to a size desirable by the user. Hence, the modular doll house may be used with traditional doll house figures or non-traditional figures such as Barbie(copyright) dolls. The modular doll house can also be easily manipulated by children to thereby ease the assembly and the disassembly of the modular doll house.
In one embodiment, the modular doll house includes a first structural member having a first surface and a first groove disposed in the first surface, a second structural member having a second surface and a second groove disposed in the second surface. Additionally, the doll house has a rigid connector including a first tab adapted to slidably engage the first groove and a second tab adapted to slidably engage the second groove to connect the first structural member to the second structural member.
In some embodiments the connector has a planar strip having first and second flat sides. A first tab may extend from the first side of the planar strip and a second tab may also extend from the first side of the planar strip and may be disposed either substantially perpendicular to or substantially parallel to the first tab. In yet another embodiment, the connector includes an L-shaped strip having first and second arms. A first tab may extend substantially perpendicularly from the first arm and may be disposed substantially parallel to the second arm of the L-shaped strip. A second tab may extend substantially perpendicularly from the second arm and may be disposed substantially perpendicularly or parallel to the first arm.
In some embodiments, the connector includes an L-shaped strip having first and second arms, a joint at the connection of the first and second arms, a planar strip and a third tab. Here, a first tab extends substantially perpendicularly from the first arm and is disposed substantially parallel to the second arm of the L-shaped strip. A second tab may extend substantially perpendicularly from the second arm and is disposed either substantially perpendicularly or parallel to the first arm. The planar strip extends substantially perpendicularly from the second arm and is fixedly attached to the second arm between the second tab and the joint for supporting a third structural member. The third tab may extend substantially perpendicularly from the planar strip and may be disposed either substantially parallel to or perpendicular to the second arm. In still another embodiment, the connector has a planar strip with two ends.
Additionally, a trough-shaped guide may be coupled to a groove in a surface of a structural member of the modular doll house, wherein the trough-shaped guide receives and slidably engages the tabs of the connector.
The modular doll house may be purchased as a kit including the structural members having surfaces adapted to receive grooves and the connectors which may have tabs to be placed in the grooves of walls, floors, ceilings, etc. to join the structural members.
Other aspects and advantages of the present invention will become apparent upon consideration of the following drawings and detailed description.